Histone deacetylase activation or expression enhancer, NAD + enhancer, and senescence cell inhibitor

Abstract

A specified sulfur-containing compound or a salt thereof, or a novel use of a composition containing the same. The present invention is a histone deacetylase activation or expression enhancing agent, NAD + enhancing agent, or senescence cell inhibiting agent containing, as an active ingredient, a compound represented by general formula 1A or 1B (however, not including S-1-propenylcysteine) or a salt thereof. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a preparation for activating or enhancing the expression of histone deacetylase, strengthening NAD+, and inhibiting senescent cells. Background Art

[0002] Sirtuins are known as molecules that extend lifespan when highly expressed in nematodes, flies, mammals, etc., and shorten lifespan when missing. Histone deacetylase is a molecule that degrades NAD. + Sir2, a histone deacetylase homolog of yeast or nematodes, shortens lifespan if it is missing, but prolongs lifespan if it is overexpressed, so histone deacetylase has attracted attention as a candidate for longevity gene in animals. There are 7 histone deacetylases (SIRT1 to 7) in mammals, and it is known that SIRT1, which is most similar to yeast Sir2 in structure and function, is likely to be related to the control of a wide range of cellular functions such as the expression of genes associated with aging, intracellular metabolism, energy consumption, inflammation and stress response pathways (Non-patent Document 1).

[0003] In addition, in recent years, it is also known that histone deacetylase may be related to specific diseases. For example, in non-patent document 2, the cognitive functions of SIRT1 knockout mice, including short-term memory, classical conditioning and spatial learning, decreased. On the contrary, it was observed that overexpression of SIRT1 would express regular synaptic plasticity and memory. It can be seen that SIRT1 plays a role in normal learning, memory, and synaptic plasticity. In addition, the correlation between hepatic steatosis and SIRT1 is recorded in non-patent document 3, and the correlation between cardiac function and SIRT1 is recorded in non-patent document 4.

[0004] Nicotinamide mononucleotide (NAD + ) functions as a coenzyme of dehydrogenase or a substrate of histone deacetylase in vivo. + Increased amounts of NAD increase the activity of histone deacetylase. The synthesis of NAD can be controlled by nicotinamide phosphoribosyltransferase (NAMPT). It is known that the role of NAMPT decreases with age, so NAD + The amount of NAD decreases, and the activity of histone deacetylase decreases. + Administration of nicotinamide riboside, a precursor of NAD, inhibits senescent cells in the brain. +, and its association with non-alcoholic fatty liver disease is recorded in Non-Patent Document 6, and its association with renal damage is recorded in Non-Patent Document 7.

[0005] Cell senescence refers to the following phenomenon, that is, excessive DNA damage is mediated by the accumulation of DNA replication errors accompanying cell division, oxidative stress, radiation, activation of oncogenes, etc., thereby activating the p16 / RB path or the p53 / p21 path, and inducing inhibitors of cell cycle protein inhibitory kinases, thereby causing irreversible cell cycle arrest. Cells that cause cell senescence (senescent cells) accumulate in tissues due to the promotion of cell senescence or the decline in the ability to remove it, and the above-mentioned cell senescence is caused by the decline in mitochondria and immune function with age. Senescent cells accumulated in tissues secrete inflammatory cytokines or proteases, which are called cell senescence-related secretory factors, thereby damaging surrounding tissues and promoting aging of tissues or organisms. On the other hand, there is a revelation that the removal of senescent cells prevents or delays the dysfunction of tissues and inhibits aging (non-patent literature 8). In addition, it is known that the increase in senescent cells is related to renal injury (non-patent literature 9) and fatty liver (non-patent literature 10).

[0006] On the other hand, it is known that sulfur-containing components existing in nature, especially in Allium plants such as garlic, have various biological functions. Examples of such components include S-alkyl (alkenyl) mercaptocysteine ​​(non-patent document 11), S-methylcysteine ​​sulfoxide (non-patent document 12), and S-allylcysteine ​​sulfoxide (non-patent document 13). However, it is known that sulfur-containing components can activate histone deacetylase or inhibit senescent cells, NAD + The enhancements are completely unknown.

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent literature 1: Trends Cell Biol. 2014; 24(8): 464-71.

[0010] Non-patent literature 2: J Neurosci 2010; 30(29): 9695-9707.

[0011] Non-patent literature 3: Molecular Medicine Reports 2018; 18: 1609-1615

[0012] Non-patent literature 4: Aging 2021; 13(10): 14482-14498

[0013] Non-patent literature 5: Cell Metab. 2018 Mar 6; 27(3): 513-528.

[0014] Non-patent literature 6: British Journal of Pharmacology 2016; 173: 2352-2368

[0015] Non-patent literature 7: Nature 2016; 531(7595): 528-532

[0016] Non-patent literature 8: Nature. 2011 Nov 2; 479(7372): 232-6.

[0017] Non-patent literature 9: Front Pharmacol. 2019; 10: 770.

[0018] Non-patent literature 10: Nature Communications 2017; 8:15691

[0019] Non-patent literature 11: J. Agric. Food Chem. 2013; 61: 1896-1903

[0020] Non-patent literature 12: Tissue Cell 2021;69:101483.

[0021] Non-patent literature 13: International Journal of Molecular Medicine 2019; 44: 1943-1951 Summary of the invention

[0022] The present invention relates to a novel use of a specified sulfur-containing compound or its salt, or a composition containing the same.

[0023] The present invention relates to the following 1) to 11).

[0024] 1) An activator or expression enhancer of histone deacetylase, NAD + An enhancer or senescent cell inhibitor, which contains a compound represented by the following general formula 1A or 1B (excluding S-1-propenylcysteine) or a salt thereof as an active ingredient.

[0025] [Chemical formula 1]

[0026]

[0027] (Where R 1 represents an alkyl group which may have a substituent, an alkenyl group which may have a substituent, or an alkynyl group which may have a substituent.2 represents a hydrogen atom or an acyl group. 3 represents a hydroxyl group or a monosubstituted amino group. X represents a group represented by -S-, -S(O)-, -S(O)2-, -SS-, -SSS- or -SSSS-. n represents 1 or 2.)

[0028] [Chemical formula 2]

[0029]

[0030] (wherein Y represents a group represented by -S- or -S(O)-).

[0031] 2) The agent as described in 1), wherein, in the general formula 1A,

[0032] R 1 represents a C1-C8 alkyl group which may have a substituent, a C2-C8 alkenyl group which may have a substituent, or a C2-C8 alkynyl group which may have a substituent,

[0033] R 2 represents a hydrogen atom, a C1-C3 alkylcarbonyl group, or a residue obtained by removing -OH from the carboxyl group of an amino acid,

[0034] R 3 It represents a hydroxyl group or a residue obtained by removing one hydrogen atom from the amino group of an amino acid.

[0035] 3) The agent as described in 2), wherein, in the general formula 1A,

[0036] R 1 represents a C1-C8 alkyl group, a hydroxy C1-C8 alkyl group, a C2-C8 alkenyl group or a C2-C8 alkynyl group,

[0037] R 2 represents a hydrogen atom,

[0038] R 3 represents hydroxyl group,

[0039] X represents a group represented by -S-, -S(O)-, or -SS-.

[0040] 4) The agent as described in 1), wherein, in the general formula 1B,

[0041] Y represents a group represented by -S-.

[0042] 5) The agent according to 1), wherein the compound is a compound represented by any one of the following structural formulas.

[0043] [Chemical formula 3]

[0044]

[0045] [Chemical formula 4]

[0046]

[0047] [Chemical formula 5]

[0048]

[0049] [Chemical formula 6]

[0050]

[0051] According to the present invention, there is provided a novel use of a predetermined sulfur-containing compound or a salt thereof, or a composition containing the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Demonstrates activation of histone deacetylase by defined sulfur-containing compounds.

[0053] Figure 2 The results show that administration of S-1-allylcysteine ​​leads to activation of histone deacetylase in the cerebral cortex.

[0054] Figure 3 The figure shows the change in the amount of SIRT1 protein in the hippocampus caused by the administration of S-1-allylcysteine.

[0055] Figure 4 Showing that administration of S-1-allylcysteine ​​leads to NAD in the hippocampus + Changes in quantity.

[0056] Figure 5 The results show that administration of S-1-allylcysteine ​​leads to changes in p53 protein, a marker of senescent cells, in the hippocampus.

[0057] Figure 6 The figure shows the change in SIRT1 expression level in the kidney caused by the administration of S-1-allylcysteine.

[0058] Figure 7 The figure shows the changes in the expression levels of p16 and p21 genes, which are senescent cell markers in the kidney, caused by the administration of S-1-allylcysteine.

[0059] Figure 8 The figure shows changes in the amount of KIM-1 and NGAL gene proteins, which are kidney injury markers, in the kidneys caused by the administration of S-1-allylcysteine.

[0060] Fig. 9 Administration of S-1-allylcysteine ​​resulted in maintenance of cognition / memory.

[0061] Fig.10 The results show that the administration of S-1-allylcysteine ​​resulted in the increase of NAD in kidney, skeletal muscle and liver + Changes in quantity.

[0062] Fig.11 The results show changes in the amount of SIRT1 protein in the cerebral cortex, hippocampus, heart, lung, liver, kidney, and skeletal muscle caused by the administration of S-1-allylcysteine.

[0063] Fig.12 The figure shows the change in the amount of SIRT1 protein in the hypothalamus caused by the administration of S-1-allylcysteine.

[0064] Fig.13 The figure shows the changes in the amount of SIRT1 protein in the liver and heart caused by the administration of S-1-allylcysteine. DETAILED DESCRIPTION

[0065] <Preparation>

[0066] The sulfur-containing compound used in the present invention is a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof. It should be noted that the sulfur-containing compound or its salt may be used alone or in combination of two or more. In the latter case, two or more single agents may be used in combination, or a combination may be used.

[0067] [Chemical formula 7]

[0068]

[0069] (Where R 1 represents an alkyl group which may have a substituent, an alkenyl group which may have a substituent, or an alkynyl group which may have a substituent. 2 represents a hydrogen atom or an acyl group. 3 represents a hydroxyl group or a monosubstituted amino group. X represents a group represented by -S-, -S(O)-, -S(O)2-, -SS-, -SSS- or -SSSS-. n represents 1 or 2.)

[0070] [Chemical formula 8]

[0071]

[0072] (wherein Y represents a group represented by -S- or -S(O)-).

[0073] R 1It may be a C1-C8 (specifically, between any two or one of C1-C8) alkyl group which may have a substituent, a C2-C8 (specifically, between any two or one of C2-C8) alkenyl group which may have a substituent, or a C2-C8 (specifically, between any two or one of C2-C8) alkynyl group which may have a substituent. Examples of the substituent include a hydroxyl group, an alkoxy group, an amino group, a carboxyl group, an aromatic group, and a heteroaromatic group.

[0074] More specifically, R 1 It can be C1-C8 (specifically, between any 2 points or 1 point of C1-C8) alkyl, hydroxy C1-C8 (specifically, between any 2 points or 1 point of C2-C8) alkyl, C2-C8 (specifically, between any 2 points or 1 point of C2-C8) alkenyl or C2-C8 (specifically, between any 2 points or 1 point of C2-C8) alkynyl.

[0075] R 2 It may be a hydrogen atom, a C1-C3 (specifically, any one or both of C1, C2, and C3) alkylcarbonyl group, or a residue obtained by removing -OH from the carboxyl group of an amino acid. Examples of the residue obtained by removing -OH from the carboxyl group of an amino acid include γ-glutaminyl group.

[0076] More specifically, R 2 It may be a hydrogen atom.

[0077] R 3 The residue may be a hydroxyl group or a residue obtained by removing one hydrogen atom from an amino group of an amino acid. Examples of the residue obtained by removing one hydrogen atom from an amino group of an amino acid include a residue obtained by removing one hydrogen atom from an amino group of glycine.

[0078] More specifically, R 3 It may be a hydroxyl group.

[0079] More specifically, X may be a group represented by -S-, -S(O)-, or -SS-.

[0080] More specifically, Y may be a group represented by -S-.

[0081] Specific examples of compounds included in Formula 1A and 1B are given below, but they are not particularly limited. It should be noted that, although not particularly limited, the compound represented by Formula 1A may be an L-type, and the compound represented by Formula 1B may be a (3R, 5S)-type.

[0082] [Chemical formula 9]

[0083]

[0084] [Chemical formula 10]

[0085]

[0086] [Chemical formula 11]

[0087]

[0088] [Chemical formula 12]

[0089]

[0090] The salt is a physiologically acceptable salt, and may be either an acid addition salt or a base addition salt. Examples of the acid addition salt include: (a) salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; (b) salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, fumaric acid, gluconic acid, malic acid, succinic acid, tartaric acid, trichloroacetic acid, and trifluoroacetic acid; (c) salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid; and examples of the base addition salt include: (a) salts with sodium, (a) salts with alkali metals such as potassium, (b) salts with alkaline earth metals such as calcium and magnesium, (c) ammonium salts, (d) salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenylethylamine, 1-diphenylhydroxymethylamine (1-Ephenamine), and N,N'-dibenzylethylenediamine.

[0091] The sulfur-containing compound or its salt may not only be in an unsolvated form, but may also exist in the form of a hydrate or a solvate, and the hydrate or solvate may exist in any crystalline form depending on the manufacturing conditions. Therefore, the sulfur-containing compound or its salt in the present invention includes all stereoisomers, hydrates, solvates, and all polymorphic forms or amorphous forms.

[0092] The sulfur-containing compound or its salt can be obtained by organic synthesis. The compound of formula 1A is not particularly limited, and can be synthesized, for example, by the following two routes.

[0093] By combining cysteine ​​or homocysteine ​​with R 1 By reacting with Br, a compound in which X is a group represented by -S- can be obtained. If it is further oxidized, a compound in which X is a group represented by -S(O)- can be obtained.

[0094] [Chemical formula 13]

[0095]

[0096] Through cysteine ​​or homocysteine ​​and R 1 -SSR 1The thiol exchange reaction can obtain a compound in which X is a group represented by -SS-. 1 -SSSR 1 Reaction, then a compound in which X is a group represented by -SSS- can be obtained. If it reacts with R 1 -SSSSR 1 The reaction can obtain a compound wherein X is a group represented by -SSSS-.

[0097] [Chemical formula 14]

[0098]

[0099] In order to get R 2 In order to obtain an acyl compound, the compound obtained above can be reacted with RCOOH or its anhydride. 3 To obtain a monosubstituted amino group, the compound obtained above may be reacted with RNH2.

[0100] The compound of formula 1B is not particularly limited and can be obtained by known synthesis methods (see, for example, Chem. Pharm. Bull. 1981; 29(6): 1554-1560).

[0101] When the sulfur-containing compound or its salt exists naturally, it can be obtained by processing its source (typically Allium plant) in various ways. Such sulfur-containing compounds or their salts are usually low in toxicity because their source (typically Allium plant) is often used as food, and are therefore preferred.

[0102] Here, as the Allium plant, garlic (Allium sativum L.), onion (Allium cepa L.), European garlic (Allium ampeloprasum L.), leek (Allium tuberosum) and shallot (Allium fistulosum L.) can be cited. These plants can be used alone or in combination. In addition, the above-mentioned Allium plants can be used fresh directly, or can be used after removing the skin and cutting or chopped as needed; or can be used as powdered materials, and can be extracted using a solvent that can be used to manufacture medicines or foods. As the solvent, water or alcohol, or a solvent obtained by adding an acid or alkaline substance to the solvent can be cited.

[0103] As the sulfur-containing compound or its salt, not only the isolated and purified one but also the crude product, the above-mentioned plant or its processed product, or the fraction in which the content of the sulfur-containing compound or its salt is increased by extraction from the above-mentioned plant can be used.

[0104] As shown in the test examples described below, various sulfur-containing compounds satisfying Formulas 1A and 1B enhance the SIRT1 activity of human cells. In addition, SIRT1 activity is enhanced in the cerebral cortex of aging-promoting mice, and SIRT1 expression is enhanced in the hippocampus, kidney, hypothalamus, heart, lung, liver and skeletal muscle. Therefore, sulfur-containing compounds, salts thereof or compositions containing them can become activation or expression enhancers of histone deacetylase or corosine.

[0105] Furthermore, NAD is used in the brain, kidney, skeletal muscle and liver of animals. + Enhance (known to enhance the function of histone deacetylase.). Therefore, sulfur-containing compounds, salts thereof or compositions containing them can become NAD + Enhancer.

[0106] In addition, cells expressing p16 and p21, which are senescent cell markers, in the kidney and cells expressing p53 in the hippocampus were inhibited. Therefore, sulfur-containing compounds, salts thereof, or compositions containing them can be senescent cell inhibitors.

[0107] If we consider the activation or enhanced expression of histone deacetylase, inhibition of senescent cells, and NAD + By strengthening the technical common sense that the mechanisms are mutually common, sulfur-containing compounds, their salts or compositions containing them can be used as histone deacetylase activation or expression enhancers, NAD + Enhancer, or senescent cell inhibitor.

[0108] The histone deacetylase activation or expression enhancer and senescent cell inhibitor of the present invention can be used to prevent, treat or improve symptoms caused by low activity or low expression of histone deacetylase, or symptoms caused by an increase in senescent cells.

[0109] As described above, the reduced function of SIRT1 and the hypersensitivity of cell senescence are said to be related to symptoms and diseases such as kidney damage, cognitive / memory impairment (including short-term and long-term), hepatic steatosis, heart failure, fatty liver, etc. (Non-patent literature 2-4, 9-10). Examples of kidney damage include: diseases with high expression of KIM-1 and NGAL, acute kidney injury, diabetic nephropathy, nephrotic syndrome, etc. Examples of cognitive / memory impairment include: neurological disorders of the cerebral cortex or hippocampus, learning disorders, memory impairment (including short-term and long-term), etc.

[0110] In the present invention, "histone deacetylase" refers to histone deacetylase proteins and homologs thereof. Among human histone deacetylases, 1 to 7 (SIRT1 to 7) are known, and in the present invention, SIRT1 is preferred.

[0111] "Enhanced histone deacetylase expression" refers to an increase in the transcription product of the histone deacetylase gene and / or histone deacetylase protein, including, for example: activation of gene transcription and / or translation, improvement of the stability of the transcription product and / or protein, inhibition of the degradation of the transcription product and / or protein, etc.

[0112] In the present invention, "cell senescence" refers to the phenomenon of irreversible cell cycle arrest in cells. Cell senescence is observed, for example, in brain cells (e.g., hippocampal cells, cerebral cortex cells), kidney cells, liver, and skeletal muscle. "Senescent cell inhibition" refers to one or both of the removal of senescent cells, the prevention or delay of cell senescence.

[0113] The p16, p21 and p53 genes are known as molecular markers of DNA damage that causes cell senescence, and the senescent cell inhibitor of the present invention preferably inhibits mitochondrial dysfunction or DNA damage that promotes cell senescence. In addition, by improving immune function, senescent cells can be more effectively inhibited.

[0114] NAD of the present invention + Increasers can be used to prevent, treat or improve NAD + Symptoms of low presence.

[0115] NAD + As mentioned above, low levels of KIM-1 are said to be associated with symptoms and diseases such as non-alcoholic fatty liver disease and renal damage (Non-patent Documents 2-4, 9-10). Examples of renal damage include diseases with high expression of KIM-1 and NGAL, acute renal damage, diabetic nephropathy, and nephrotic syndrome.

[0116] NAD of the present invention + The enhancer, histone deacetylase activation / expression enhancer, and senescent cell inhibitor may be in the form of a drug or food, or may be in the form of a material or preparation added thereto.

[0117] In addition, this food contains: NAD + Foods labeled as strengthening, histone deacetylase activation / expression enhancement or senescent cell inhibition and explaining the effects based on the functions as necessary, functional foods, functional labeled foods, foods for patients, foods for specific health purposes, and nutritional supplements (supplements).

[0118] The dosage form of the drug is preferably a dosage form suitable for oral administration. Specific dosage forms of oral administration preparations include, for example, tablets, capsules, fine granules, pills, and granules as solid preparations; emulsions, solutions, suspensions, syrups, and the like as liquid preparations. The drug preparation can be prepared by appropriately mixing excipients, binders, disintegrants, lubricants, colorants, flavor correctors, pH adjusters, and the like with the sulfur-containing compound or its salt of the present invention as needed, and prepared according to conventional methods.

[0119] However, the dosage form of the drug is not particularly limited, and may be a dosage form suitable for parenteral administration, such as a dosage form suitable for intravenous administration (injection, catheter), transmucosal administration (liquid or ointment), or intracranial administration (catheter).

[0120] The form of the food is not particularly limited, and may be, for example, solid food, semi-fluid food, gel food, tablets, capsule-type tablets, capsules, and more specifically, may be snacks, beverages, seasonings, processed seafood, processed meat, bread, health food, and other forms of food.

[0121] The food can be produced by appropriately mixing food materials commonly used in producing such food with the sulfur-containing compound or its salt of the present invention and following a conventional method.

[0122] The above medicines or foods may contain NAD + Other substances related to enhancement, activation of histone deacetylase, expression enhancement or inhibition of senescent cells, such as resveratrol or nicotinamide mononucleotide. In addition, it can contain vitamins, lipids, and minerals that relieve inflammation, such as vitamin C, vitamin E, vitamin B2, vitamin B6, niacin, hesperidin, α-lipoic acid, glutathione, coenzyme Q10, zinc, magnesium, ω-3 fatty acids, etc.

[0123] The preferred daily intake of the above-mentioned medicine or food varies depending on factors such as the object of intake, the form of intake, the type of materials or additives taken at the same time, the intake interval, etc. As for the sulfur-containing compound or its salt, it is preferably taken 0.001 to 10 mg / kg per day, and more preferably 0.1 to 1 mg / kg per day. In addition, the daily amount can also be taken in 2 to 4 times as needed. It should be noted that the amount of the sulfur-containing compound or its salt here can be the amount of one kind or the total amount of two or more kinds.

[0124] Examples of the subjects for administration or ingestion include decreased activity or expression of histone deacetylase, increased senescent cells, or NAD +The organism may be an organism with a reduced amount of leukemia, but may also be a healthy organism without these or specific diseases (e.g., diabetes, kidney damage). The organism is not limited to the animals described above, but also includes: plants, fungi (including yeast), nematodes, cells (which may be derived from the above animals or plants.), but preferably animals. Examples of the animal include: vertebrates (preferably rodents and primates), fish, birds, insects and reptiles, and rodents (especially rats and mice) and primates (especially humans and monkeys) are particularly preferred.

[0125] As far as the inventors know, there is no evidence that the sulfur-containing compounds of Formula 1A and 1B, when administered to animals, can cause histone deacetylase activation or expression enhancement, NAD + These are the results of in-depth research conducted by the inventors and others, and are unexpected findings.

[0126] Examples include humans suffering from renal damage, cognitive / memory impairment (including short-term and long-term), hepatic steatosis, heart failure, fatty liver, non-alcoholic fatty liver disease, and animals (e.g., humans) for whom prevention of such diseases is desired, but healthy organisms are also preferred.

[0127] According to the suggestion, histone deacetylase in plants is related to genomic instability or protecting cells from oxidative damage, gamete formation, fruit development and ripening, leaf senescence, and regulation of photosynthetic activity (Front Plant Sci. 2018 Jul 5; 9: 961.).

[0128] Plants are not particularly limited, and examples thereof include: tomatoes, bell peppers, peppers, eggplants and other solanaceous vegetables; cucumbers, pumpkins, melons, watermelons and other melons; celery, parsley, lettuce and other lettuce-spice vegetables; onions, onions, garlic and other onions; beans such as soybeans, peanuts, green beans, peas, red beans and other beans; other fruits and vegetables such as strawberries; taproots such as radishes, turnips, carrots, burdocks and other tapioca roots; taro such as taro, cassava, potato, sweet potato, long taro and other taro; asparagus, spinach, duck celery and other tender vegetables. Soft vegetables; flowers such as Turkish lisianthus, violets, carnations, chrysanthemums, etc.; cereals such as rice and corn; grasses such as sedge and Korean grass; oil crops such as rapeseed and sunflower seeds; sugar crops such as sugarcane and beets; fiber crops such as cotton and rush; fodder crops such as clover, sorghum, and dent corn; deciduous fruit trees such as apples, pears, grapes, and peaches; citrus fruits such as Wenzhou mandarin, lemon, and grapefruit; woody plants such as camellia, azalea, and fir.

[0129] The dosage form for administration to plants includes powders, granules, pellets, hydrates, flowables, emulsions, and pastes. The drug may be administered directly to the plant or indirectly via soil, hydroponic solution, or culture medium. Specifically, soil treatment agents, stem and leaf treatment agents, seed treatment agents before sowing, treatment agents for plants before transplantation, treatment agents for plants during transplantation, hydroponic solution, culture medium, and the like may be mentioned.

[0130] <Other methods>

[0131] In other embodiments, the present invention may also relate to a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof, or a composition containing a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof, which is used for the above-mentioned various applications.

[0132] In other embodiments, the present invention may also relate to a method for administering a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof, or a composition containing a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof for the above-mentioned various applications.

[0133] In other embodiments, the present invention may also relate to the use of a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof, or a composition containing a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof in the above-mentioned various applications.

[0134] In other embodiments, the present invention may also relate to the use of a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof, or a composition containing a compound represented by the general formula 1A or 1B (however, excluding S-1-propenylcysteine) or a salt thereof in the manufacture of an agent for the above-mentioned various uses.

[0135] The details of these means are the same as those of the above-mentioned agents.

[0136] Example

[0137] Reference Synthesis Example (S-1-Propylene Cysteine)

[0138] Dissolve tert-butoxypotassium in dimethyl sulfoxide, add S-allylcysteine, and stir for 10 minutes. Move to an ice bath, and add 12M hydrochloric acid under vigorous stirring. Next, add ethanol and filter out the resulting crystals. Recrystallize the resulting crystals with 30% ethanol to obtain S-1-allylcysteine.

[0139] Synthesis Example 1 (S-propylcysteine: SPC)

[0140] Dissolve cysteine ​​in water, add bromopropane ethanol solution, and stir. Add triethylamine dropwise to the mixed solution, and stir overnight at room temperature. After concentration, add ethanol, and filter out the precipitated crystals. After washing the crystals with ethanol, recrystallize with water solvent to obtain S-propylcysteine.

[0141] Synthesis Example 2 (5-Methylthiomorpholine-3-carboxylic acid: Cyclo-SAC)

[0142] According to the method of Chem. Pharm. Bull. 1981; 29(6): 1554-1560, 5-methylthiomorpholine-3-carboxylic acid was obtained.

[0143] Synthesis Example 3 (S-3-butenylcysteine: SBenC)

[0144] S-3-butenylcysteine ​​was obtained according to the method of Synthesis Example 1 except that bromopropane was changed to bromobutylene.

[0145] Synthesis Example 4 (S-2-Hydroxypropylcysteine: S2HyPC)

[0146] Dissolve cysteine ​​in a mixed solvent of methanol and water and stir. Add 28% ammonia water in an ice bath and drop 1-bromo-2-propanol methanol solution. After 10 minutes of stirring, return to room temperature and stir overnight. Extract the reaction solution with chloroform and concentrate the aqueous layer. Purify the crystals obtained after concentration using HPLC to obtain S-2-hydroxypropylcysteine.

[0147] Synthesis Example 5 (S-methylmercaptocysteine: SMMC)

[0148] Dimethyl disulfide and hydrogen peroxide are added to acetic acid and stirred. Next, cysteine ​​is added and stirred. Ammonia water is used for neutralization, and the precipitated crystals are washed with water and methanol. The crystals are dissolved in 1N hydrochloric acid and washed with diethyl ether. Then, ammonia water is added for neutralization, and the precipitated crystals are washed with water and methanol to obtain S-methylmercaptocysteine.

[0149] Synthesis Example 6 (S-methylcysteine ​​sulfoxide (Methiin))

[0150] Add methyl bromide to a 2N ammonia solution and stir. Next, add cysteine ​​and stir further. After concentrating the solution, recover the resulting crystals. Wash the resulting crystals with water and ethanol to obtain S-methylcysteine. Oxidize the S-methylcysteine ​​with hydrogen peroxide, and recrystallize the resulting crystals with a mixed solvent of acetone and water. Wash the precipitated crystals with acetone to obtain S-methylcysteine ​​sulfoxide.

[0151] Synthesis Example 7 (S-ethylcysteine ​​sulfoxide (Ethiin))

[0152] Add ethyl bromide to a 2N ammonia solution and stir. Next, add cysteine ​​and stir further. After concentrating the solution, recover the resulting crystals. Wash the resulting crystals with water and ethanol to obtain S-ethylcysteine. Oxidize the S-ethylcysteine ​​with hydrogen peroxide, and recrystallize the resulting crystals with acetone and water. Wash the precipitated crystals with acetone to obtain S-ethylcysteine ​​sulfoxide.

[0153] Synthesis Example 8 (Alliin)

[0154] Add allyl bromide to a 2N ammonia solution and stir. Next, add cysteine ​​and stir further. After concentrating the solution, recover the produced crystals. Wash the obtained crystals with water and ethanol to obtain S-allylcysteine. Oxidize the S-allylcysteine ​​with hydrogen peroxide, and recrystallize the obtained crystals with acetone and water. Wash the precipitated crystals with acetone to obtain S-allylcysteine ​​sulfoxide.

[0155] Test example

[0156] 50 μM of trichostatin A and 10 mM of dithiothreitol were added to 200 ng of human cervical cancer cell lysate and incubated at 37°C for 30 minutes to promote endogenous NAD + Next, each sulfur-containing compound synthesized above was added at a concentration of 3 mM, and after being placed at 37°C for 10 minutes, an enzyme reaction was performed using Cyclex SIRT1 / Sir2Deacetylase Fluorometric Assay Kit Ver.2 (manufactured by the Institute of Medical Biology) to measure the histone deacetylase activity. The results are shown in Figure 1 Any sulfur-containing compound will enhance the activity of histone deacetylase ( Figure 1 ).

[0157] Reference Example 1 Ethanol Extract of Garlic

[0158] About 1 kg of garlic bulbs with the skin removed and about 1000 mL of 30% ethanol are placed in a sealed container. The container is placed at room temperature for 1 to 10 months with appropriate stirring. Solids and liquids are separated from the mixture, and the liquid is dried by spray drying to obtain a yellow-brown powder.

[0159] Reference Preparation Example 2 Isolation of S-1-Propylene Cysteine ​​from Ethanol Extract of Garlic

[0160] (1) The ethanol extract fraction of garlic obtained in Preparation Example 1 was placed in a dialysis tube with a pore size of 3500 and dialyzed against purified water. The dialyzed external solution was passed through a cation exchange resin Dowex50Wx8 (H+), and the resin was well washed with purified water. The amino acids adsorbed on the resin were eluted with 2N ammonia and concentrated under reduced pressure. The concentrate was attached to a silica gel column and subjected to column chromatography using a chloroform / methanol / water mixture as a solvent. The fraction containing the target substance (S-1-propenylcysteine) was recovered and concentrated. The concentrate was dissolved in water, chromatographed using a preparative reverse phase column (octadecylsilane column) with 0.1% formic acid as a solvent, the target substance was recovered, and the solvent was removed by freeze drying. The structure of the obtained freeze-dried product was compared with the spectrum obtained from the standard substance shown below by NMR (solvent: heavy water) and mass spectrometry, and it was confirmed that it was a mixture of trans-S-1-propenylcysteine ​​and cis-S-1-propenylcysteine ​​(trans isomer: cis isomer = 8:2).

[0161] trans-S-1-allylcysteine

[0162] 1 H-NMR(500MHz,in D2O-NaOD, δ): 1.76 (d, 3H, J = 7.0Hz), 2.98 (dd, 1H, J = 7.5, 14.5Hz), 3.14 (dd, 1H, J = 4 .5,14.5Hz)3.69(dd,1H,J=4.5,7.5Hz),5.10-5.14(m,1H),6.02(d,1H,J=15.5Hz);

[0163] 13 C-NMR(125MHz,in D2O-NaOD,δ):17.61,33.53,53.70,119.92,132.12,172.73,

[0164] HRMS: observed[M+H] + =162.0583, calculated [M+H] + =162.0581

[0165] cis-S-1-Propylene Cysteine

[0166] 1 H-NMR(500MHz,in D2O, δ): 1.74 (d, 3H, J = 7.0Hz), 3.21 (dd, 1H, J = 7.5, 15.0Hz), 3.31 (dd, 1H, J = 4.5 ,15.0Hz),3.95(dd,1H,J=4.5,7.5Hz),5.82-5.86(m,1H),6.01(d,1H,J=9.5Hz);

[0167] 13 C-NMR (125MHz, in D2O-NaOD, δ): 13.89, 33.88, 54.16, 122.58, 127.78, 172.63.

[0168] HRMS: observed[M+H] + =162.0580, calculated [M+H] + =162.0581

[0169] (2) Determination of S-1-allylcysteine ​​in ethanol extract of garlic

[0170] 500 mg to 1 g of the ethanol extract of garlic obtained in Production Example 1 (1) was placed in a container, and a 20 mM hydrochloric acid solution of Sn-3-butenylcysteine ​​was added as an internal standard, and the solution was made up to 20 mL with 20 mM hydrochloric acid. After sufficient stirring, a portion was taken and centrifuged at 1750 G for about 10 minutes. A portion of the obtained supernatant was taken and centrifuged (15000 rpm, 10 minutes) using a centrifugal filter unit (Amicon Ultra, cutoff: 3000). 20 μL of the obtained filtrate was taken and derivatized using the AccQ·Tag Derivatization Kit (Waters). In addition, the standard compound was dissolved in 20 mM hydrochloric acid, and the same operation as the sample was performed to prepare a standard solution for the calibration curve. The sample solution and the standard solution were chromatographed using an Acquity UPLC system (Waters) to determine the content. As a result, the content of S-1-butenylcysteine ​​was 3.7±0.3 mg / g dry matter.

[0171] Reference test example

[0172] Histone deacetylase activation

[0173] (1) Sample preparation

[0174] Preparation of the test solution for evaluating biological activity was performed as follows: When evaluating biological activity, the test solution was prepared before use.

[0175] (a) About 5 mg of S-1-propenylcysteine ​​(cis / trans mixture) produced in Production Example 2 was accurately weighed and dissolved in 10 mL of purified water to prepare a dosing solution.

[0176] (b) About 6 mg of S-1-propenylcysteine ​​(cis / trans mixture) produced in Production Example 2 was accurately weighed and dissolved in 1 mL of culture medium. This solution was used as a stock solution and appropriately diluted for use in an in vitro test.

[0177] (2) Evaluation of experimental animals

[0178] Animals used for evaluation of biological activity were bred as follows: Experimental accelerated aging mice SAMP8 (male) were purchased from Japan SLC and acclimated for one week after purchase to be used as evaluation test animals.

[0179] (3) Preparation of human neuroblasts for evaluation experiments

[0180] Human neuroblastoma cell line SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotic penicillin and streptomycin solution, and the obtained cells were used as evaluation test cells.

[0181] (4) Histone deacetylase activation assay

[0182] In order to measure the activity of histone deacetylase, a SIRT1_GLO kit manufactured by Promega was purchased. The cerebral cortex solution was placed in a 96-well plate, and after adding various reaction reagents, the luminescence intensity of luciferase was measured using a plate reader.

[0183] (a) SIRT1 activation (in vivo):

[0184] The test animals of the evaluation method of (2) were used to orally administer a single dose of the sample prepared in (1) (a) above, and then the cerebral cortex was removed after 15, 30, 60, and 180 minutes. The cerebral cortex was crushed using an automatic tissue crusher and then dissolved by adding a cell lysate. The cells were centrifuged and the activity of histone deacetylase in the supernatant was measured. The results are shown in Figure 2 .

[0185] 180 minutes after administration of S-1-allylcysteine, histone deacetylase activity was enhanced. Figure 2 )

[0186] (b) SIRT1 protein increase effect (in vivo):

[0187] Using the evaluation test animals of (2) above, the sample prepared in (1) (a) above was repeatedly orally administered for 2 weeks, and then the hippocampus was removed. The hippocampus was crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with a protease / phosphatase mixed inhibitor manufactured by ThermoFisher Scientific. The cells were centrifuged (10000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to a conventional method. Antibodies used were anti-SIRT1 antibody (manufactured by Biolegend) and anti-β-actin antibody (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The results are shown in Figure 3 S-1-allylcysteine ​​increased SIRT1 protein amount.

[0188] (C)NAD + Volume-increasing effect (in vivo):

[0189] Using the test animals for evaluation of (2), the sample prepared in (1) (a) was orally administered once, and then the cerebral cortex was removed 180 minutes later. Also, using the test animals for evaluation of (2), the sample prepared in (1) (a) was mixed with the feed, and the feed was fed for 1 month, and then the cerebral cortex was removed. NAD in the cerebral cortex + NAD produced by Tongren Chemical Research Institute + / NADH Assay Kit was used for determination. The results are shown in Figure 4 . S-1-Propylenecysteine ​​increases NAD + quantity.

[0190] (5) Inhibition of p53 protein expression, a marker of cellular senescence, in the hippocampus (in vivo):

[0191] Using the evaluation test animals of (2) above, the sample prepared in (1) (a) above was repeatedly orally administered for 6 weeks, and then the hippocampus was removed. The hippocampus was crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with protease inhibitors and phosphatase inhibitors manufactured by Roche. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to a conventional method. Antibodies used were anti-p53 antibody (manufactured by Proteintech) and anti-β-actin antibody (manufactured by the Institute of Medical Biology). The results are shown in Figure 5 S-1-allylcysteine ​​reduced the amount of p53 protein.

[0192] (6) Enhancement of SIRT1 gene expression in kidney (in vivo):

[0193] Using the evaluation test animals described in (2) above, the sample prepared in (1) (a) above was repeatedly orally administered for 2 weeks, and then the kidneys were removed. The kidneys were crushed using an automatic tissue crusher, and RNA was extracted using a TRIzol solution manufactured by Thermo Fisher Scientific. cDNA was synthesized using the PRIMEScript RT reagent Kit with gEraser manufactured by TAKARA. Real-time polymerase chain reaction (Real-Time PCR) was performed on the synthesized cDNA using the KAPASYBR Fast qPCR kit manufactured by NIPPON Genetics. The results are shown in Figure 6 S-1-allylcysteine ​​enhanced the expression of SIRT1 gene in mouse kidney.

[0194] (7) Inhibition of p16 and p21 gene expression, markers of senescent cells in the kidney (in vivo):

[0195] The test animals of the evaluation method of (2) were repeatedly orally administered with the sample prepared in (1) (a) for 2 weeks, and then the kidneys were removed. The kidneys were crushed using an automatic tissue crusher, and RNA was extracted using TRIzol solution manufactured by Thermo Fisher Scientific.

[0196] cDNA was synthesized using PRIMEScript RT reagent Kit with gEraser manufactured by TAKARA. Real-time PCR was performed on the synthesized cDNA using KAPASYBR Fast qPCR Kit manufactured by NIPPON Genetics. The results are shown in Figure 7 S-1-allylcysteine ​​reduces p16 and p21 gene-expressing cells in the kidneys of senescent mice.

[0197] (8) Kidney damage inhibition effect (in vivo)

[0198] Using the evaluation test animals of the above (2), the sample prepared in the above (1)(a) was repeatedly orally administered for 2 weeks, and then the kidneys were removed. The kidneys were crushed using an automatic tissue crusher, and the cells were lysed with RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with protease inhibitors and phosphatase inhibitors manufactured by Roche, and then centrifuged (10000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. Using this cell extract, analysis was performed by protein blotting according to a conventional method. Antibodies used were kidney injury molecule (KIM-1) antibody (manufactured by R&D system) and Lipocalin-2 (NGAL) antibody (manufactured by Proteintech), which are kidney injury markers, and anti-GAPDH antibody (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The results are shown in Figure 8 S-1-Propylenecysteine ​​reduced KIM-1 and NGAL.

[0199] (9) Cognitive / memory maintenance effect (in vivo):

[0200] Using the evaluation test animals of (2) above, the sample prepared in (1) (a) above was mixed into the bait, and after feeding for 4 months, a passive avoidance test was conducted. The passive avoidance test is a test that utilizes the habit of mice to prefer darker places. If the mice placed in the bright room move to the dark room, they will be subjected to electrical stimulation (50V·1 second). The time before the mice move to the dark room on the first day is used as the reaction latency of the collection test. After 2 months, the mice are placed in the bright room again, and the time to move to the dark room (the reaction latency of the retention test) is used as the evaluation method of the memory pointer. The results are shown in Fig. 9 S-1-allylcysteine ​​improves cognitive / memory decline due to ageing.

[0201] (10) NAD in kidney, skeletal muscle and liver + Volume-increasing effect (in vivo):

[0202] The test animals of the evaluation method of (2) were orally administered once with the sample prepared in (1) (a) above, and then 60 minutes later, the cerebral cortex, liver, kidney, and skeletal muscle were removed. NAD in cerebral cortex, liver, kidney, and skeletal muscle + NAD produced by Tongren Chemical Research Institute + / NADH Assay Kit was used for determination. The results are shown in Fig.10 . S-1-Propylenecysteine ​​increases NAD + quantity.

[0203] (11) Increased SIRT1 protein in the cerebral cortex, hippocampus, heart, lung, liver, kidney, and skeletal muscle (in vivo):

[0204] About 10 mg of S-1-propenylcysteine ​​(cis / trans mixture) produced in Production Example 2 was accurately weighed and dissolved in 10 mL of purified water to prepare a dosing solution.

[0205] Using the evaluation test animals of (2) above, the prepared sample was repeatedly orally administered for 2 weeks, and then the cerebral cortex, hippocampus, heart, lung, liver, kidney, and skeletal muscle were removed. The cerebral cortex, hippocampus, heart, lung, liver, kidney, and skeletal muscle were crushed using an automatic tissue crusher, and then lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with a protease / phosphatase mixed inhibitor manufactured by Thermo Fisher Scientific. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to a conventional method. Antibodies used were anti-SIRT1 antibody (manufactured by Biolegend) and anti-β-actin antibody (manufactured by Fuji Film Wako PureChemical Industries, Ltd.). The results are shown in Fig.11 S-1-allylcysteine ​​increased SIRT1 protein amount.

[0206] (12) Increased effect of SIRT1 protein in the hypothalamus (in vivo):

[0207] S-1-propenylcysteine ​​(cis / trans mixture) produced in Production Example 2 was dissolved in pure water at a concentration of 0.001% to prepare a dosing solution.

[0208] Animals used for evaluation of biological activity were bred as follows: Experimental aging-promoting mice SAMR1 (male) and SAMP8 (male) were purchased from Japan SLC. After purchase, they were acclimated for one week and used as evaluation test animals.

[0209] The evaluation test animals were fed with normal food or, for SAMP8, the sample prepared above was mixed with the feed, and the hypothalamus was removed after feeding for 10 months. The hypothalamus was crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with a protease / phosphatase mixed inhibitor manufactured by Thermo Fisher Scientific, and then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to a conventional method. Antibodies used were anti-SIRT1 antibody (manufactured by Biolegend) and anti-β-actin antibody (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The results are shown in Fig.12 S-1-allylcysteine ​​increased SIRT1 protein amount.

[0210] (13) Increased SIRT1 protein levels in the liver and heart (in vivo):

[0211] The sample prepared in (1) (a) was repeatedly orally administered to the evaluation test animals prepared in the same manner as (12) for 2 weeks, and then the liver and heart were removed. The liver and heart were crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with a protease / phosphatase mixed inhibitor manufactured by Thermo Fisher Scientific. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to a conventional method. Antibodies used were anti-SIRT1 antibody (manufactured by Biolegend) and anti-β-actin antibody (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The results are shown in Fig.13 S-1-allylcysteine ​​increased SIRT1 protein amount.

Claims

1. An activator or expression enhancer of histone deacetylase, NAD + An enhancer or senescent cell inhibitor, which contains a compound represented by the following general formula 1A or 1B or a salt thereof as an active ingredient, but the compound represented by the general formula 1A or 1B does not include S-1-propenylcysteine; In the formula, R 1 represents an alkyl group which may have a substituent, an alkenyl group which may have a substituent, or an alkynyl group which may have a substituent; R 2 represents a hydrogen atom or an acyl group; R 3 represents a hydroxyl group or a monosubstituted amino group; X represents a group represented by -S-, -S(O)-, -S(O)2-, -SS-, -SSS- or -SSSS-; n represents 1 or 2; In the formula, Y represents a group represented by -S- or -S(O)-.

2. The agent according to claim 1, wherein In general formula 1A, R 1 represents a C1-C8 alkyl group which may have a substituent, a C2-C8 alkenyl group which may have a substituent, or a C2-C8 alkynyl group which may have a substituent, R 2 represents a hydrogen atom, a C1-C3 alkylcarbonyl group, or a residue obtained by removing -OH from the carboxyl group of an amino acid, R 3 It represents a hydroxyl group or a residue obtained by removing one hydrogen atom from the amino group of an amino acid.

3. The agent according to claim 2, wherein In general formula 1A, R 1 represents a C1-C8 alkyl group, a hydroxy C1-C8 alkyl group, a C2-C8 alkenyl group or a C2-C8 alkynyl group, R 2 represents a hydrogen atom, R 3 represents hydroxyl group, X represents a group represented by -S-, -S(O)-, or -SS-.

4. The agent according to claim 1, wherein In general formula 1B, Y represents a group represented by -S-.

5. The agent according to claim 1, wherein The compound is a compound represented by any one of the following structural formulas;

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